Detection systems for registering protein interactions and functional relationships

ABSTRACT

Disclosed herein are methods for detecting complex protein interactions and protein functional relationships, and reagents for carrying out those methods.

This application claims benefit of the filing date of provisionalapplication, U.S. Ser. No. 60/065,273, filed Nov. 10, 1997, nowabandoned

BACKGROUND OF THE INVENTION

This invention relates to protein interaction detection systems.

Genetic analysis is a tool for understanding the protein networks thatgovern biological processes. The manipulations performed by geneticists(e.g., staging of temperature sensitive mutants, construction andanalysis of double mutants, and generation and observation of F1 and F2progeny) define relationships between genes. These abstractrelationships between genes often reflect underlying biologicalrealities. For example, the epistasis relation may suggest that one genenormally acts on another to cause a phenotype, the allelic specificsuppression relation may suggest that two gene products physicallyinteract (Hartman and Roth, Adv. Genet. 17:1-105 (1973); Jarvik andBotstein, Proc. Natl. Acad. Sci. USA 70:2046-50 (1973)), and thedependency relation may suggest that the action of one gene productprecedes that o<another in time (Hereford and Hartwell, J. Mol. Biol.84:445-61 (1974)).

Information obtained from these genetic manipulations is typically ofvery high quality, but is often relatively difficult to acquire. Therecent increase in the rate of identification of new coding sequenceshas renewed interest in global systematic methods to understand genefunction. These methods include the “two hybrid” or “interaction trap”methods which have been developed to assay contact between a single baitand interacting proteins (Fields and Song, Nature 340:245-6 (1989);Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-82 (1991); Gyuris etal., Cell 75:791-803 (1993); Durfee et al., Genes Dev. 7:555-69 (1993);Estojak et al., Mol. Cell. Biol. 15:5820-9 (1995). Contact between twoproteins in these systems defines a physical relationship that isfrequently of biological significance.

SUMMARY OF THE INVENTION

In general, the invention features a method for detecting aprotein—protein interaction, involving: (a) providing a host cell whichcontains (i) a first reporter gene operably linked to a DNA sequencethat includes a first protein binding site; (ii) a second reporter geneoperably linked to a DNA sequence that includes a second protein bindingsite; (iii) a first fusion gene which expresses a first fusion protein,the first fusion protein including a first protein covalently bonded toa binding moiety which is capable of specifically binding to the firstprotein binding site; (iv) a second fusion gene which expresses a secondfusion protein, the second fusion protein including a second proteincovalently bonded to a binding moiety which is capable of specificallybinding to the second protein binding site; and (v) a third fusion genewhich expresses a third fusion protein, the third fusion proteinincluding a third protein covalently bonded to a gene activating moiety;(b) measuring expression output of the first reporter gene as a measureof the interaction between the first and the third proteins; (c)measuring expression output of the second reporter gene as a measure ofthe interaction between the second and the third proteins; and (d)interpreting the expression output results of step (b) and step (c),whereby (i) increased output of both of the first and the secondreporter genes indicates that the third fusion protein interacts withboth of the first and the second fusion proteins; (ii) increased outputof the first reporter gene but not the second reporter gene indicatesthat the third fusion protein interacts with the first fusion proteinbut not the second fusion protein; (iii) increased output of the secondreporter gene but not the first reporter gene indicates that the thirdfusion protein interacts with the second fusion protein but not thefirst fusion protein; and (iv) no change in output in either of thefirst or the second reporter genes indicates that the third fusionprotein does not interact with either of the first or the second fusiongenes.

In a preferred embodiment, the method further involves comparing theexpression output results of step (b) and step (c) with the expressionoutput result measured in either (a) a first comparison host cell whichcontains (i) a reporter gene operably linked to a DNA sequence includinga protein binding site; (ii) a first fusion gene which expresses a firstfusion protein, the first fusion protein including the first proteincovalently bonded to a binding moiety which is capable of specificallybinding to the protein binding site; and (iii) a second fusion genewhich expresses a second fusion protein, the second fusion proteinincluding the third protein covalently bonded to a gene activatingmoiety; or (b) a second comparison host cell which contains (i) areporter gene operably linked to a DNA sequence including a proteinbinding site; (ii) a first fusion gene which expresses a first fusionprotein, the first fusion protein including the second proteincovalently bonded to a binding moiety which is capable of specificallybinding to the protein binding site; and (iii) a second fusion genewhich expresses a second fusion protein, the second fusion proteinincluding the third protein covalently bonded to a gene activatingmoiety; or (c) both of the first and the second comparison host cells.In addition, any number of comparisons may be made between multiple“two-bait” cells or between a “two-bait” cell and multiple “one-bait”cells, or both.

In other preferred embodiments, at least one of the first or the secondreporter genes may be reduced in expression level; one of the first orthe second protein binding sites is a tetracycline operator; one of thefirst or the second reporter genes is URA3 or lacZ; one of the first orthe second reporter genes produces a signal that is received anddetected by a second cell; the host cell is a yeast cell or a mammaliancell; the first and the second reporter genes may be expressedsimultaneously; and the first protein and the second proteins areallelic variants.

In a second aspect, the invention features a method for detecting aprotein that mediates a change in the state of another protein,involving: (a) providing a host cell which contains (i) a reporter geneoperably linked to a DNA sequence including a protein binding site; (ii)a first fusion gene which expresses a first fusion protein, the firstfusion protein including a first protein covalently bonded to a bindingmoiety which is capable of specifically binding to the protein bindingsite; and (iii) a second fusion gene which expresses a second fusionprotein, the second fusion protein including a second protein which iscapable of interacting with the first protein and which is covalentlybonded to a gene activating moiety, wherein at least one of the first orthe second proteins may exhibit a change in state; (b) allowing thefirst and the second proteins to interact; (c) measuring expression ofthe reporter gene as a measure of the interaction between the first andthe second proteins; (d) introducing into the cell a third geneexpressing a third protein; (e) measuring expression of the reportergene, a change in the reporter gene expression in the presence of thethird protein being an indication that the third protein mediates achange in the state of the first or the second protein leading to analteration in the ability of the first protein and the second protein tointeract.

In a related aspect, the invention features an alternative method fordetecting a protein that mediates a change in the state of anotherprotein, involving: (a) providing a first host cell which contains (i) areporter gene operably linked to a DNA sequence including a proteinbinding site; (ii) a first fusion gene which expresses a first fusionprotein, the first fusion protein including a first protein covalentlybonded to a binding moiety which is capable of specifically binding tothe first protein binding site; and (iii) a second fusion gene whichexpresses a second fusion protein, the second fusion protein including asecond protein which is capable of interacting with the first proteinand which is covalently bonded to a gene activating moiety, wherein atleast one of the first or the second proteins may exhibit changes instate; (b) allowing the first and the second proteins to interact; (c)measuring expression of the reporter gene in the first host cell as ameasure of the interaction between the first and the second proteins;(d) providing a second host cell which contains (i) the reporter geneoperably linked to the DNA sequence including the protein binding site;(ii) the first fusion gene which expresses the first fusion protein; and(iii) the second fusion gene which expresses the second fusion protein;(iv) a third gene which expresses a third protein; (e) allowing thefirst and the second proteins to interact in the presence of the thirdprotein; (f) measuring expression of the reporter gene in the secondhost cell as a measure of the interaction between the first and thesecond proteins in the presence of the third protein, a change in thereporter gene expression in the second host cell as compared to thatmeasured in the first host cell being an indication that the thirdprotein mediates a change in the state of the first or the secondprotein resulting in an alteration in the ability of the first proteinand the second protein to interact.

In preferred embodiments of each of the above methods, the change instate is a conformational change; the protein exhibiting aconformational change is a Ras protein; the host cell is a yeast cell ora mammalian cell; the expression of the first fusion protein and thethird protein occurs in response to an extracellular stimulus; and thereporter gene produces a signal that is received and detected by asecond cell;.

In another related aspect, the invention features a cell that includes(i) a first reporter gene operably linked to a DNA sequence including afirst protein binding site; (ii) a second reporter gene operably linkedto a DNA sequence including a second protein binding site; (iii) a firstfusion gene which expresses a first fusion protein, the first fusionprotein including a first protein covalently bonded to a binding moietywhich is capable of specifically binding to the first protein bindingsite; (iv) a second fusion gene which expresses a second fusion protein,the second fusion protein including a second protein covalently bondedto a binding moiety which is capable of specifically binding to thesecond protein binding site; and (v) a third fusion gene which expressesa third fusion protein, the third fusion protein including a thirdprotein covalently bonded to a gene activating moiety.

In preferred embodiments, at least one of the first or the secondreporter genes may be reduced in expression level; one of the first orthe second protein binding sites is a tetracycline operator; one of thefirst or the second reporter genes is URA3 or lacZ; one of the first orthe second reporter genes produces a signal that is received anddetected by a second cell; and the host cell is a yeast cell or amammalian cell.

In yet another related aspect, the invention features a reporter genethat includes a tetracycline operator operably linked to a gene encodinga detectable product (for example, a URA3 gene or a lacZ gene).

In a final aspect, the invention features a method for detecting whethera candidate protein interacts with a transcriptional activator,involving: (a) providing a host cell which contains (i) a reporter genewhich can be reduced in expression level, the reporter gene beingoperably linked to a DNA sequence including a protein binding site;(iii) a first fusion gene which expresses a first fusion protein, thefirst fusion protein including the transcriptional activator covalentlybonded to a binding moiety which is capable of specifically binding tothe protein binding site; and (v) a second fusion gene which expresses asecond fusion protein, the second fusion protein including the candidateprotein covalently bonded to a gene activating moiety; (b) detecting anincrease in expression of the reporter gene as an indication of aninteraction between the candidate protein and the transcriptionalactivator.

In preferred embodiments, the reporter gene is a URA3 gene; theexpression level is reduced by 6-azauracil; the protein binding site isa tetracycline operator; the expression level is reduced by tetracyclineor a tetracycline derivative; and the host cell is a yeast cell or amammalian cell.

By a “reporter gene” is meant a nucleic acid sequence whose expressionmay be assayed; such genes include, without limitation, lacZ, amino acidbiosynthetic genes, and the mammalian chloramphenicol transacetylase(CAT) gene. Reporter genes may be assayed, for example, by a change incell viability or color reaction.

By “expression output” is meant any measurable change in reporter geneexpression.

By a “protein binding site” is meant a nucleic acid sequence that may berecognized and bound by a protein or peptide.

By a “transcriptional activator” is meant an amino acid sequence that iscapable of increasing expression of a gene to which it is bound. A “geneactivating moiety” is all or a portion of such a transcriptionalactivator that is capable of increasing gene expression.

By a “tetracycline derivative” is meant a compound that is related totetracycline and that is capable of inhibiting the binding of atetracycline repressor to a tetracycline operator. An exemplarytetracycline derivative is anhydrotetracycline.

By “a change in state” is meant any physical or chemical change in aprotein that alters its activity. Examples of changes in state includechanges in phosphorylation patterns or conformation.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The drawings will first be described.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C is a series of three panels which schematically illustratean exemplary two-bait interaction trap of the invention. In Cell 1 (FIG.1A), on glucose medium, a transcriptionally-inert tetracycline repressor(TetR) fusion (Bait1, Ba₁) and a LexA fusion (Bait2, Ba₂) are bound torespective Tet and LexA operators upstream of reporters. The potentiallyinteracting protein, fused to the transcription activation domain B42 isnot expressed (Gyuris et al., Cell 75:791-803 (1993)). In this system,neither reporter gene is activated, the cell grows on 5-fluorooroticacid (5-FOA) medium and does not grow on uracil⁻ medium, and is white on5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal) medium. In Cell 2(FIG. 1B), on galactose and raffinose medium, Prey 1, a protein fused toB42, is expressed, but does not interact with either bait. The reportersare not activated, the cell grows on 5-FOA or does not grow on uracil⁻medium, and is white on Xgal. In Cell 3 (FIG. 1C), on galactose andraffinose medium, another prey, Prey 2, is expressed and interacts withBa₁, but not Ba₂. In this cell, the URA3 reporter is selectivelyexpressed, the cell grows on uracil⁻ medium but is white on Xgal.

FIG. 2 is a table of results showing cells that register the logical“And” and discrimination relationships (“Ls1” and “Ls2”) on interactionrelationships A₁ and A₂. Cells contain the indicated bait and preyproteins; preys are only expressed on galactose and raffinose medium.Reporter output is indicated by growth or blue color on the indicatedmedium. For the And relationship, the prey protein contacts both baitsand activates both reporters (TetOp-URA3 and LexOp-LacZ). For the Ls1relationship, the prey interacts only with the LexA fusion bait, andactivates only the LexOp-lacZ reporter. Similarly, in the Ls2relationship, the prey contacts only the TetR fusion bait, and activatesonly the TetOp-URA3 reporter.

FIGS. 3A-3C are a series of results showing cells that register thelogical And operation on input proteins. FIG. 3A is a table showingexpression of the LexOp-LacZ reporter in CWXY2 cells 1 and 2; expressionis indicated by the intensity of the blue color on LHWK/Gal+Raff+Xgalplates (see below) and by their β-galactosidase activity (indicated inMiller units). FIG. 3B is a schematic illustration of two cells witheither TetR-Cdc25 (907-1589) or TetR-GAP, on Gal+Raff+Xgal medium (seebelow). In Cell 1 on the Gal+Raff plate, the TetR-Cdc25 loadsLexA-RasA₁₈₆ with GTP, allowing binding of more B42-c-Raf1 and increasedtranscription of the LexAOp-lacZ reporter. In Cell 2, TetR-GAPstimulates Ras GTPase activity so that more LexA-Ras is in the GDP-form,resulting in less B42-c-Raf1 binding, and decreased transcription of theLexAOp-LacZ reporter. FIG. 3C is a truth table depicting the results ofoperations on protein inputs. In this figure, “1” in the second columndenotes respectively the presence of TetR-Gap or TetR-Cdc25, and “0” or“1” in the third column denotes respectively a low or a high output ofβ-galactosidase.

FIG. 4 is a schematic illustration of a cell that produces an outputthat may be read by the producer cell or a second, recipient cell.

FIG. 5 is a schematic illustration of a cell having four input-outputchannels.

FIG. 6 is a schematic illustration of a wholly biological circuit thatacts as a transistor.

As described herein, the present invention provides methods and reagentsfor detecting or registering complex relationships between two or moreproteins. Many of these relationships, for example, “bridging” (orconnecting) and “discriminating” interactions, are useful forunderstanding gene function. As demonstrated below, by performinglogical operations on the phenotypic outputs of both new and existingtwo-hybrid cell interactions, detailed models of protein function inpathways and complexes can be determined. The protein relationshipsdetected by these cells are analogous to classical genetic relationshipssuch as epistasis, but they can be interpreted within a rigorousanalytical framework, and they can be performed systematically on theproducts of entire genomes. In addition, cells that register suchrelationships can perform logical operations on protein inputs, and thusmay define paths for the construction of biological computationaldevices.

Preliminary Considerations: Logical Protein Relationships in Two-HybridSystems

In classical (“one bait”) two-hybrid systems, the output of a reportergene depends on the interaction between the DNA-bound bait andactivation-tagged prey. Genetic markers expressed by some reporters, forexample, URA3, allow selection against reporter transcription and thusfor lack of interaction (Le Douarin et al., Nucl. Acids Res. 23:876-8(1995)). The relationship between proteins in these systems can bedescribed in symbolic-logical terms.

By this view, contact between a bait (Ba₁) and a prey (P₁) defines avariable (A₁), here called a contact relationship. Because A₁ isoperationally defined by the reporter output, the term A₁ is used torefer to the reporter output as well. There are four possible Booleanoperations, or functions, on this relationship (Schneeweiss, BooleanFunctions: With Engineering Applications and Computer Programs(Springer-Verlag, Berlin, N.Y., 1989)). Two of these are constantfunctions: F1 (A₁)=0 and F2 (A₁)=1, and two are true functions: F3(A₁)=A₁ and F4 (A₁)=Not A.

In one-bait systems, the phenotypic consequences of the reporter outputcan register two of these operations on the contact relationship, F3 andF4. This is shown in Table 1. In this Table, the contact relationshipbetween the Bait Ba₁ and the Prey P₁ is denoted as A₁, and is measuredby the output of the reporter. Thus, the values of A1 (0 and 1) refer toboth the values of the contact relationship and to the off and on statesof the reporter. “Value of A₁” shows the two possible values (0 and 1 oroff and on) of A1. “Operation” shows the two allowed operations(functions) on A₁. The two subcolumns of “Results of operation” refer tothe output of the operations on A₁. “Alternative names” gives commonnames for the operations. “Interpretation” describes the state ofinteraction between the bait and prey proteins. “Defining phenotype”gives the phenotype for a contact relationship in the one bait system,and “Example biological correlates” gives examples of the biologicalcircumstances that can result in such outputs.

In Table 1, both F3 and F4 have important biological correlates.Consider a one bait system, in which contact between Ba₁ and P₁ (forexample, because these proteins heterodimerize) gives a positive output(blue color on X-Gal, growth on Ura⁻ medium), while loss of that contact(for example, by mutation or disruption by a peptide aptamer) (Colas etal., Nature 380:548-550 (1996)) gives a negative output (white color onX-Gal, growth on 5-FOA medium) (Table 1). A cell growing on 5-FOA mediumis analogous to a device that performs the logical Not operation on thecontact relationship, or, alternatively, as a cell that registers thestate (Not A₁).

Cells that Detect More Complex Protein Relationships

Cells were constructed that allowed simultaneous selection for andagainst two distinct protein—protein interactions (FIGS. 1A-1C). Thistwo-bait interaction trap utilized the DNA binding moieties of LexA andTetR, the tetracycline repressor of bacterial transposon Tn10 (Gossenand Bujard, Proc. Natl. Acad. Sci. 89:5547-51 (1992)). The LexA and TetRfusion bait proteins were expressed in a yeast cell that also containedan integrated TetOp-URA3 reporter and an episomal LexAOp-lacZ reporter.Expression of baits in these “two-bait” cells was under the control ofthe ADH1 promoter. The TetOp-URA3 reporter was integrated into the LYS2gene. The LexAOp-lacZ reporter, a derivative of pSH18-34, was carried onpCXW24, a 2 μm plasmid with a LYS2 marker. The prey vector was pJG4-5,whose GAL1 promoter conditionally directed expression of anactivation-tagged protein from a 2 μm TRP1 plasmid (Gyuris et al., Cell75:791-803 (1993)).

Logical Operations on a Contact Relationship in a Two-Bait InteractionTrap

In the two-bait cells, the two contact relationships (and the output ofthe corresponding reporters) were expressed as Boolean variables, A₁ andA₂. There were 16 possible operations on these variables (Schneeweiss,Boolean Functions: With Engineering Applications and Computer Programs(Springer-Verlag, Berlin, N.Y., 1989)), four of which were registered inthese cells. These operations were referred to as And, Nor, and the twodiscrimination operations, logic state 1 (Ls1) and logic state 2 (Ls2).And, Ls1, and Ls2 were considered to represent particularly usefuloperations for determining protein function. A summary of theseoperations is shown in Table 2. In that Table, the contact relationshipbetween Ba₁ and P₁ is denoted as A₁, and is measured by the output ofthe TetOp-URA3 reporter. The contact relationship between Ba₂ and P₁ isdenoted as A₂, and is measured by the output of the LexAop-lacZreporter. Again, values A₁ and A₂ (0 and 1) are used to refer to boththe values of the contact relationship and to the values (off and onstates) of the reporter. The four subcolumns of “Value of variables”denote the four different possible combinations of values for the twovariables. “Operation” shows the four operations on these variablespossible in this system. “Alternative names” gives common names for theoperations. “Interpretation” describes the state of interactions betweenthe bait and prey proteins. “Defining phenotype” gives the phenotype fortouching relationship in the one bait system, and “Example biologicalcorrelates” gives examples of the biological circumstances that canresult in such outputs.

To test the general utility of the system, a set of illustrativeproteins were used, called RasA₁₈₆, RasA₁₅A₁₈₆, RasV₁₂A₁₈₆, GAP,hSos1(residues 601-1019), Cdc25 (residues 907-1589), c-Raf1 (residues1-313), Max, and Mxi1. The Harvey ras gene product, Ras, is a GTPasethat exists in two distinct conformations, a GDP-bound (inactive) formand a GTP-bound (active) form. The fact that Ras cycles between theseconformations allows it to be a switch protein in signal transductionpathways that control cell proliferation (Boguski and McCormack, Nature366:643-654 (1993)). All Ras proteins described herein contained a Cysto Ala change at 186, which inactivated a farnesylation site andincreased the apparent nuclear concentration of the protein. RasA₁₈₆exists in a mixture of GDP and GTP bound forms, while RasA₁₅A₁₈₆ andRasV₁₂A₁₈₆ are predominantly in the GDP and GTP bound forms,respectively. GAP, which stimulates GTPase activity of Ras, binds toGTP-bound Ras. c-Raf1, a downstream target of Ras, also binds GTP-boundRas. By contrast, hSos1 and Cdc25, both of which activate Ras, only bindto GDP-bound Ras. Max and Mxi1, which heterodimerize tightly, were usedas positive controls (Zervos et al., Cell 79:388 (1993)).

FIG. 2 shows that the two-bait cell registered the logical Andoperation. The relationship is fulfilled by proteins, such as bridgingproteins in pathways, that can interact with both baits. In thisexperiment, yeast strain CWXY2 carried B42-RasA₁₈₆ as prey, TetR-c-Raf1and LexA-hSos1 as the baits, and TetOp-URA3 and LexAOp-LacZ as thereporters. B42-RasA₁₈₆ interacted with both TetR-c-Raf1 and LexA-hSos1.The cell was blue on Xgal and grew on medium lacking uracil. Thisbridging or connecting relationship was consistent with the idea thatRas interacted with both proteins, and suggested that proteins thatactivate both reporters can be selected from genome-wide screens.

In addition, FIG. 2 shows that the two-bait system also registered Ls1and Ls2, the discrimination relationships. These relationships areexpected for proteins that interact with one bait but not another. Here,in cells expressing TetR-RasV₁₂A₁₈₆ and LexA-Max baits and a B42-c-Raf1prey, the Ras-Raf interaction activated the TetOp-URA3 reporter, and thecells grew in medium lacking uracil; Raf did not interact with LexA-Maxand thus did not activate the lacZ reporter. On the other hand, in acell expressing TetR-RasV₁₂A₁₈₆ and LexA-Max baits and a B42-Mxi1 prey,the Max-Mxi1 interaction activated the LexAop-LacZ reporter, and thecells turned blue on Xgal; Mxi1 did not interact with Ras and thus didnot activate the URA3 reporter.

Moreover, the results in FIG. 2 indicated that these cells coulddiscriminate between two closely related allelic variants. In a cellexpressing TetR-RasA₁₅A₁₈₆, LexA-RasV₁₂A₁₈₆, and B42-c-Raf1, theRaf/RasV₁₂A₁₈₆ interaction activated expression of the LexAop-LacZreporter; the cells turned blue on Xgal but did not grow on mediumlacking uracil. By contrast, in a cell expressing TetR-RasA₁₅A₁₈₆,LexA-RasV₁₂A₁₈₆, and B42-Cdc25, the Cdc25/RasA₁₅A₁₈₆ interactionactivated the TetOp-URA3 reporter, and allowed the cells to grow onmedium lacking uracil but caused it to remain white on Xgal. This resultsuggested that these cells could identify, from genomic or combinatoriallibraries, proteins and peptides that interact with allelic proteinvariants specific for disease states.

Identification of Peptide Aptamers That Discriminate Between AllelicVariants

A two-bait cell that contained TetR-RasV₁₂ and LexA-RasA₁₅ was used toisolate members of a peptide aptamer library that interactedspecifically with RasV₁₂. URA⁺ library transformants were screened forlacZ⁻ cells, which presumably contained aptamers that did not interactwith LexA-RasA₁₅. Plasmids encoding aptamers were then rescued fromthese lacZ⁺ cells and their phenotypes reconfirmed. Using this system,two discriminatory aptamers, Pep22 and Pep104, were identified. Pep22interacted with both RasV₁₂ and RasA₁₅, whereas, by contrast, Pep104interacted only with RasV₁₂. In particular, the Pep22-containing cellgrew on Ura⁻ medium and was blue on X-gal medium. The Pep104-containingcell grew on Ura⁻ medium but was white on X-gal medium. These resultsdemonstrated the utility of this system in selection of specific peptideaptamers. For Pep22, the second bait increased the selectivity of thesystem by eliminating potential false positives that might arise fromartifactual activation of a single reporter. For Pep104, the second baitallowed detection of aptamers specific for an allelic form of theprotein active in signal transduction. The sequences of Pep22 and Pep104are DMDWFFRFYA SVSRLFRHLH (SEQ ID NO: 15) and FWQATLRLVS DKLILLYPDP (SEQID NO: 16), respectively.

Logical Operations on Protein Inputs

Cells can also perform logical operations on protein inputs, andregister the result of those operations by changes in output. FIGS.3A-3C show a logical And operation on protein inputs. In a cellexpressing LexA-RasA₁₈₆, B42-c-Raf1, and TetR-GAP, the output of theLexAop-lacZ reporter was low (light blue on Xgal medium), presumablybecause GAP drove most of the Ras to the GDP-bound conformational state.By contrast, input of TetR-Cdc25 increased the RasA₁₈₆/c-Raf1interaction, as shown by the intensity of blue color on Xgal plates,presumably by changing Ras into the Raf binding conformation. In thisexperiment, the cell had two inputs, one of which, B42-c-Raf1 (logicalvalue 1) was constantly present, while the other was either TetR-GAP(logical value 0) or TctR-Cdc25 (logical value 1); and the output waseither high (1) or low (0). In this case, the output of the LexAop-lacZreporter was thus controlled by a LexA-Ras switch protein whoseconformation depended on the values of the inputs.

In an alternative to the above system, Cdc25 may be replaced with hSos1,and similar results obtained. In addition, expression of systemcomponents may be further regulated. For example, in the systemsdescribed above, B42-c-Raf1 may be expressed using a GAL1 promoter,and/or Cdc25 and hSos1 may be expressed from a promoter normallyrepressed by TetR-Sin3. This results in expression of B42-c-Raf1 beingdependent on the presence of galactose in the growth medium, andexpression of Cdc25 and hSos1 being dependent on the presence oftetracycline or a tetracycline derivative in the growth medium. In thesesystems, the cell is responding to two distinct extracellular inputs,one that controls the expression of the modifying protein and the otherthat controls expression of the prey.

Protein Relationships in Two Hybrid Systems

Based on the experiments described above, the outputs of reporter genesin one bait two-hybrid systems may be viewed as reflecting two basiclogical states. The activation of the reporter gene embodies the contactrelationship A₁ between the bait 1 (Ba₁) and prey (P₁). Not A₁ (betweenBa₁ and P₁) defines the lack of interaction, caused, for example, by amutation in the interacting partners, or disruption of the interactionby a third protein or peptide aptamer (Colas et al., Nature 380:548-550(1996)).

Construction of Cells with Independently Functional InteractionReporters

As described above, cells were constructed that detected more complexprotein relationships by making a version of the interaction trap thatutilized the DNA binding moieties of LexA and TetR, the tetracyclinerepressor of bacterial transposon Tn10. Fusion proteins containing thesemoieties were expressed as two baits in a cell that also containedTetOp-URA3 and LexAop-lacZ reporters. This system allowed simultaneousdetermination of two genetic interactions in a single cell.

The inclusion of TetR baits and TetOp-URA3 reporters significantlyfacilitated interaction trap applications. The phenotype dependent onthe TetOp-URA3 reporter was more sensitive than that of lacZ and LEU2reporters (see, for example, Gyuris et al., Cell 75:791-803 (1993) andEstojak et al., Mol. Cell. Biol. 15:5820-9 (1995)) which facilitatesdetection of weak interactions. In addition, both the URA3 and LacZreporter genes may be quantitatively assayed (Shostak et al., Anal.Biochem. 191:365-9 (1990)). Moreover, the sensitivity of this URA3reporter can be down-regulated in two ways. Expression of the URA3reporter can be titrated by 6-azauracil, an inhibitor of the URA3 geneproduct (orotidine-5′-monophosphate decarboxylase (OMPdecase) (LeDouarin et al., Nucl. Acids Res. 23:876-8 (1995). Reporter activity canalso be reduced by tetracycline or its derivatives (for exampleanhydrotetracycline), which disrupt binding of the TetR bait to Tetoperators. Preferably, such compounds are used at a concentration of upto 100 μg/ml on plates. Both kinds of inhibitors diminish thesensitivity of the URA3 reporter, allowing its use with baits thatactivate transcription and allowing its use, along with lacZ, for crudeestimation of interaction affinities. Moreover, the URA3 reporter allowsthe use of 5-FOA to select against gene expression (Boeke et al., Mol.Gen.Genet. 197:345-6 (1984)). In this case, tetracycline and 6-azauracilcan regulate the threshold amount of transcription selected against,facilitating the selection of peptide aptamers that break specificprotein interactions.

Logical Analysis of Binary and Higher-Order Protein Relationships

As shown in Table 2, the two bait cells registered four logicalrelationships, Nor, And, Ls1, and Ls2. Three are particularly important.The And relationship (A₁ (between Ba₁ and P₁) and A₂ (between Ba₂ andP₁) was found for prey proteins (connecting proteins) that contactedboth baits (Table 2). Identification of such proteins is useful forcontinued construction of dense charts of genetic networks and forconnecting pathways not previously known to be related. Such sets ofinteracting proteins are sometimes referred to As “protein contigs.”

Ls1 and Ls2, the discrimination relationships, were also important.These relationships are relatively complex: for example, the Ls1relationship involves two operations on two interactions: Not A₁(between Ba₁ and P₁) and A₂ (between Ba₂ and P₁). These operations havenumerous biological correlates, in that a cell that registers thisrelation allows detection of proteins that interact differently withunrelated proteins, allelic variants, and different conformationalstates (FIG. 2), and also allows detection of proteins that interactdifferentially with different modification states. The use of theserelationships to survey the products of combinatorial libraries andgenomes allow selection of proteins that interact specifically withproteins encoded by disease-state alleles, or with proteins that differfrom wild-type due to differential splicing or posttranslationalmodification.

Analysis of Higher-Order Protein Relationships

The protein relationships that can be inferred from two-bait cells arenot always identical to those revealed by one-bait cells. For example,if both Ba₁ and Ba₂ oligomerize to form a surface that interacts with P₁then neither the Ba₁/P nor Ba₂/P interaction will be detected inone-bait cells. Such differences in contact relationships are useful,since combining data from one- and two-bait cells allows theexperimenter to make inferences about the topology, temporal sequence,and posttranslational modification dependent of the proteininteractions.

Table 3 shows inferences about physical interactions among threeproteins, X, Y, and Z: (i) from possible outputs of a two-bait cell thatdetects contact relationships A₁ (between X and Z), A₂ (between Y andZ), and A₃ (between X and Y). In this Table, “reporter output” shows thestate of the contact relationships registered by outputs of thereporters in three one-bait cells and a single two-bait cell. X and Zare fused to an activation domain to form preys [P(X) and P(Z)], and Yand X are fused to a DNA binding domain to form baits [Ba(X) and Ba(Y)].In a two-bait cell, the outputs of the reporters show the state of thecontact relationship for proteins X, Y, and Z where they are fused withone of two DNA binding domains [Ba₁(X) or Ba₂(Y)] and an activationdomain [P(Z)]. “Physical interpretation” shows one possible biologicalinterpretation of this set of reporter outputs for combinations of one-and two-bait data, or for one-bait data alone. Although all patterns inTable 3 may not have biological correlates, many have been observed, forexample, the interaction of Bait₁ and prey depends on the presence ofBait₂. Experiments such as those depicted in Table 3 indicating linkagesof one- and two-bait data are useful in ordering the function ofproteins in pathways and complexes.

Application to the Analysis of Gene Function

This two-bait system, particularly when combined with existing one baitsystems, thus extends the scope of yeast interaction technology toanalyze the function of genes in pathways. It can aid the identificationof proteins and peptide aptamers that distinguish between allelicvariants of proteins. In addition, linkage of data from two-bait cells(likely to result from individual experiments) and from one-bait cells(perhaps obtained from genome-wide surveys) allows detailed analysis ofprotein function and contact topology in pathways and complexes. It alsoallows more precise analysis of the topology of proteins inmulti-protein complexes, and it provides robust, scalable,semi-automatic ways to distinguish among alternative models of proteininteractions. In addition, because the relationships among proteinsdefined in this way lend themselves to systematic analysis, they can bedetermined industrially.

Towards Protein-and-Transcription Based Logical Circuitry

The above experiments made use of a protein, Ras, that cycles betweentwo conformational states, and an activation tagged protein, Raf, thatbinds Ras in one of these states. The state of the Ras switch, and itsoutput measured by transcription, was shown to vary, depending onwhether the input protein was GAP or Cdc25. In these experiments thecells were acting as And gates, in which one input, B42-c-Raf1, was heldconstant (logical value 1), the other was either Gap (logical value 0)or Cdc25 (logical value 1), and the phenotypes caused by expression ofthe reporters constituted the outputs. With B42-c-Raf1 and GAP, theoutput was low β-galactosidase activity (0). With B42-c-Raf1 and Cdc25,the output was high β-galactosidase activity (1). Although it was alsoscored by reporter expression, this And operation was not an operationon the contact relationship like those described earlier. Rather, theoperation was on the protein inputs, and in this case the cells wereacting as true logic gates.

In the cells described herein, to change the inputs, different DNAconstructions were employed that expressed interacting proteins; tomeasure output, human or other observers were needed. Construction ofwholly biological transcription-based logic circuitry requires replacingthese inputs and outputs with logical inputs that vary in response toextracellular stimuli, such as secreted peptide pheromones or light, andoutputs that generate such stimuli.

Exemplary systems employing such biological circuitry are shown in FIGS.4-6. In particular, in FIG. 4, a cell is depicted that produces anoutput that may be read by the producer cell or by a second, recipientcell in response to the input. As shown in FIG. 4, addition of the inputprotein, yeast α-factor, results in the expression, through a G proteinpathway, of a TGF-β output, an output that may be received by the sameor a second cell and that may produce a phenotypic change in therecipient cell. FIG. 4 also depicts a system in which a TGF-β inputprotein binds to its receptor, resulting in the activation andtranslocation of a LexA-Madd (also referred to as a LexA-Smad) protein.This LexA-Madd protein triggers expression of a yeast α-factor output, asignal that again may be read by the producer cell or by a second,recipient cell and may produce a phenotypic change in the recipientcell.

FIG. 5 extends this system and illustrates a cell having four differentinput-output channels, thereby allowing for a variety of logicaloperations. In FIG. 5, the input proteins α-factor, TGF-β, delta, andbradykinin interact with the α-factor receptor, TGF-β receptor, notchreceptor, and bradykinin receptor, respectfully, to activate pathwaysthat result in expression of output proteins that may be read bycellular systems, for example, as transcriptional changes. As the numberof input and output options increases, the number of channels that maybe programmed increases; for example, a cell having 10 input/outputchannels may be programmed in 2¹⁰, or more than 1000 different states.In one exemplary approach to increasing input/output channels, aprotein, such as the LexA-Smad protein discussed above, may beengineered to contain different aptameric moieties, each conferring adifferent receptor specificity on the chimera. Moreover, in thesesystems, the input, rather than being a protein, may be some otherextracellular stimuli, such as light or a particular wavelength of lightthat triggers a specific photoreceptor pathway. Preferably, in such asystem, the output is a fluorescent protein, for example, a green, red,or blue fluorescent protein.

One final type of wholly biological circuit is depicted in FIG. 6. Thiscell represents an exemplary “transistor.” In this cell, in the presenceof green light, a reporter gene is triggered to express an HIV protease.This protease then cleaves a target linker that joins together a bluefluorescent protein-green fluorescent protein chimera, releasing bothcomponents, and resulting in a large decrease in green light output.

The wholly biological logical devices described above might not be veryfast. Although one of these switch proteins, Ras, can cycle inmilliseconds and a number of signal transduction pathways can provideinputs within minutes (Bray, Nature 376:307-312 (1995)), reporter outputmay require minutes to hours to be detectable. However, because requiredconstruction work near the DNA may be performed using straightforwardtechniques, it is likely that gene expression will remain a usefuloutput. Construction of such circuits is facilitated by the rapidincrease in the number of natural and artificially selected proteindomains with useful allosteric and targeting properties (Colas et al.,Nature 380:548-550 (1996)). It is thus possible that suchtranscription-based technologies provide an early route to biologicalcomputation.

Material and Methods

The Two-Bait System

In the experiments described above, the vectors pEG202 and pJG4-5 wereutilized as Ba₂ and prey expression plasmids (Gyuris et al., Cell75:791-803 (1993)). In addition, Ba₁ expression plasmid, pCWX200,LexAOp-LacZ reporter plasmid, pCWX24, and TetOp-URA3 reporter vectorswere constructed and were integrated into Saccharomyces cerevisiaestrains to create CWXY1 and CWXY2 as follows.

Construction of the TetR-fusion protein expression vector, pCWX200.pCWX200 carried a 2 μm replicator and LEU2 marker. The promoter andcloning region derived from pEG202 and the backbone from pBC100, aderivative of Yeplac 181 (Gietz and Sugino, Gene 74:527-534 (1988)). Toremove the pBC100 polylinker region (from AccI to EcoRI) as well as DNAencoding the amino-terminal fragment of β-galactosidase, the vector wasdigested with NarI/AccI and religated to yield pCWX100. The HindIII sitewas then deleted by digesting pCWX100 with HindIII, blunt-ending theoverhangs with Klenow, and religating to yield pCWX100ΔHindIII. The SphIfragment of pEG202, containing the ADH1 promoter, LexA, a polylinker,and the ADH1 terminator was then inserted into the SphI site ofpCWX100ΔHindIII to yield pCWX150 (Gyuris et al., Cell 75:791-803 (1993)and Estojak et al., Mol. Cell. Biol. 15:5820-9 (1995)). Finally, theEcoRI/HinIII LexA fragment of pCWX150 was replaced with an EcoRI/HindIIIended PCR fragment that encoded the Tet repressor (TetR) (Zervos et al.,Cell 79:388 (1993)). The resulting plasmid, pCWX200, had cloning sitesequivalent to those of pEG202 (Gossen and Bujard, Proc. Natl. Acad. Sci.89:5547-51 (1992)).

Construction of the LexAop-lacZ reporter, pCWX24. The LexAop-lacZreporter, the LYS2 marker, the 2 μm replicator, and the rest of thepCWX24 plasmid backbone were derived from intermediate plasmids pCWX221,pCWX01, and pCWX21, respectively.

Construction of pCWX221. To construct this plasmid, SH18-34T wasdigested to completion with KpnI, a partial digestion with BamHI wasperformed, and the vector was ligated to a BamHI/KpnI digested backboneof pBluescript. The LexAOp-LacZ reporter was on the NotI/KpnI fragmentof pCWX221.

Construction of pCWX01. To construct pCWX01, YeP426 was digested withSalI/ClaI (Ma et al., Gene 58:201-16 (1987)), and a ˜5600 bp ClaI-XbaIfragment was obtained that carried LYS2 (Barnes and Thorner, Mol. Cell.Biol. 6:2828-38 (1986)) and about 300 bp of the pBR322 tetracyclineresistance gene. This fragment was ligated with the (pBluescriptΔKpnI)pBSΔKpnI backbone digested with ClaI/SalI, to generate pCWX01.

Construction of pCWX21. pRF24 was digested with NotI and blunt-endedwith Klenow to yield pCWX20ΔNotI. To create pCWX21, pCWX20ΔNotI wasdigested with BamHI and ligated to self-annealed 5′-GATCCGCGGCCGCG-3′(SEQ ID NO: 1) to generate a new NotI site.

Assembly of pCWX24. To assemble pCWX24 NotI/KpnI LexOp-LacZ fragment ofpCWX221 was inserted into the NotI/KpnI site of pCWX21 to create pCWX22.Next, a self-annealed 5′-CTAGGGCCCTAGCATG-3′ fragment was inserted atthe SphI site of pCWX22 to generate an ApaI site and the vector pCWX23.After digesting pCWX23 with ApaI/PmeI, a SmaI/ApaI digested LYS2fragment of pCWX01 was inserted to create pCWX24.

Construction of TetOp-URA3. The TetOp-URA3 constructs were generatedfrom the intermediate plasmids, pCWX211, pCWX213T2, pCWX213T10,pCWX02T2, and pCWX02T10.

Construction of pCWX211. Following amplification of the URA3 gene frompSH200 with 5′-AAAAGGAAAAGCGGCCGCTTAGTTTTGCTGGCCGCATCTTC-3′ (SEQ ID NO:2) and 5′-CGGAATTCTTTCGAAAGCTACATATAAGGAAC-3′ (SEQ ID NO: 3), theNotI/EcoRI ended PCR product was ligated to EcoRI/NotI-digested pBS togenerate p(CWX211.

Construction of pCWX213T2 and pCWX213T10. To construct these vectors, aBamHI fragment from pLR1Δ1, containing the yeast Gal1/Gal10 promoterregion with a unique XhoI site (West et al., Mol. Cell Biol. 4:2467-78(1984)), was inserted into the BamHI site of pBS to create pCWX210. Twostrands were then synthesized and annealed to create an XhoI/SalI-endedDNA fragment that contained two TetO₂ operators (Meier et al., EMBO J.7:567-72 (1988)), as follows (SEQ ID NOS: 4 and 5):

                  *                           *           TCGAGcactccctatcagtgatagagaaaacactccctatcagtgatagagaaaaG1−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−−−−+−−−−−−56      CgtgagggatagtcactatctcttttgtgagggatagtcactatctcttttCAGCT

After ligating the Tet operator DNA to itself, it was digested withXhoI/SalI and fractionated on a 0.8% agarose gel. DNA with sizes rangingfrom 100 bp to 200 bp was inserted at the XhoI site of pCWX210. Usingthis protocol, two plasmids, pCWX210T2 and pCWX210T10, were obtained,which contained 4 and 6 TetO₂ operators respectively. The EcoRI-endedGal1/Gal10 promoter regions of pCWX210T2 and T10 were inserted into theEcoRI site of pCWX211 to generate pCWX211T2 and pCWX211 T10respectively. In addition, a KpnI site in these plasmids was created byinserting a self-annealed oligo (5′-GGCCGCGGTACCGC-3′) (SEQ ID NO: 6) atthe NotI site to create pCWX212T2 and pCWX212T10 respectively. In thesevectors, the TetOp-URA3 reporter was positioned on a KpnI fragment.Deletion of the ClaI site in these plasmids produced plasmids, pCWX213T2and pCWX213T10. Replacement of the KpnI fragment of LYS2 from pCWX01with KpnI-ended TetOp-URA3 constructs from pCWX213T2 and pCWXT10 createdpCWX02T2 and pCWX02T10.

Integration of the TetOp-URA3 Reporter at LYS2. Following digestion ofpCWX02T2 and pCWX02T10 with ClaI/XbaI, these vectors were transformedinto Saccharomyces cerevisiae strain EGY40 containing pCWX200Cdi2 (Gietzet al., Yeast 11:355-60 (1995)), a vector which directed expression ofthe TetR-Cdi2 gene, a transcription activator (Finley and Brent, Proc.Natl. Acad. Sci. 91:12980-84 (1984)). Transformants were selected onmedium lacking uracil and leucine. Ten independent clones were pickedfor each pCWX02T2 and pCWX02T10 transfomation, and streaked them ontoalpha-aminoadipic acid plates to select for those in which LYS2 wasinactivated (Chattoo et al., Genetics 93:51-65 (1979)). Integration ofthe TetOp-URA3 gene at the LYS2 locus was confirmed by PCR using primerswhich hybridized to the regions flanking the KpnI fragment of LYS2. Theresulting strains CWXY1 and CWXY2 (MATa ura3 his3 trp1 leu2lys2Δkpn::TetOp-URA3) contained 4 and 6 TetO₂ operators upstream of theURA3 gene, respectively.

Assays for the And and Discrimination Relationships

To assay interaction relationships, a series of vectors wereconstructed. c-Raf1 (1-313) was cloned into the EcoRI/XhoI sites ofvectors pCWX200 and JG4-5. Following amplification of RasA₁₅A₁₈₆,hSos1(601-1019), and Cdc25(907-1589) with RasA₁₅A₁₈₆ primers5′-GCCTGAATTCATGACGGAATATAAGCTGG-3′ (SEQ ID NO: 7) and5′-CCCGAACTCGAGTCAGGAGAGCACTGCCTTGCAGC-3′ (SEQ ID NO: 8), the hSos1(601-1019) primers 5′-GCCTGAATTCAAAGCAGGAACTGTT-3′ (SEQ ID NO: 9) and5′-CCCGAACTCGAGCTATCGTGGTTCTATTTCTAG-3′ (SEQ ID NO: 10), and the Cdc25catalytic domain (907-1589) primers 5′GCCTGAATTCATGTCTTCGGTCTCCTCAG-3′(SEQ ID NO: 11) and 5′-CCCGAACTCGAGTTATCGAAATAACCTAGAAGG-3′ (SEQ ID NO:12), the EcoRI/XhoI-ended PCR products of RasA₁₅A₁₈₆, hSos1(601-1019),and Cdc25(907-1589) were also cloned into the EcoR1/Xho1 sites ofvectors pEG202 and pJG4-5. Having transformed CWXY2 containing pCWX24with the combinations of two baits and preys as above in FIG. 2,transformants were streaked onto dropout plates lacking leucine,histidine, lysine, and tryptophan (“LHKW”), having glucose as a carbonsource (“Glu”), and supplemented with 100 μg/ml 6-Azaruracil, aninhibitor of URA3 (Le Douarin et al., Nucl. Acids Res. 23:876-8 (1995)).These plates were incubated at 30° C. for 12-48 hours. Yeast streakswere next replicated onto the four dropout plates, LHKW/Glu+X Gal,LHKW/Galactose (Gal)+Raffinose (Raff)+X Gal, LHKWU/Glu, andLHKWU/Gal+Raff as shown above, and the results were scored afterincubation at 30° C. for 12-72 hours.

Assays for the And Operation on Protein Inputs

To carry out additional assays, an EcoRI/XhoI-ended PCR product of humanGAP1, amplified with primers 5′-GCCTGAATTCATGAAGGGGTGGTATCACGGA-3′ (SEQID NO: 13) and 5′-CCCGAACTCGAGCTA-CTTGACATCATTGGTTTTTG-3′ (SEQ ID NO:14), was cloned into pCWX200. EcoRI/XhoI-ended Cdc25(907-1589) (as inFIG. 2) was also cloned into pCWX200. CWXY2 containing pEG202RasA₁₈₆ andpJGRaf(1-313) was then further transformed with either pCWX200,pCWX200CDC25(907-1589), or pCWX200GAP. Streaks of transformants pooledfrom >50 independent transformants were replicated onto LHKW/Glu+Xgaland LHKW/Ga1+Raff+Xgal dropout plates, and these plates were incubatedat 30° C. for 2-3 days. Cells on the former plate showed no blue color,and, on the latter plate, showed blue color of varied intensity.β-galactosidase activity was measured by liquid assays in triplicates ofcell pools (Estojak et al., Mol. Cell. Biol. 15:5820-9 (1995)), each ofwhich contained >50 independent transformants. After inoculatingLHKW/Gal+Raff medium at an OD₆₀₀ of 0.2 with washed cells grown to latelog phase or saturation in LHKW/Glu liquid media, cultures wereincubated at 30° C. for an additional 5 hours, and β-galactosidaseassays were performed as described in Estojak et al. (Mol. Cell Biol.15:5820-29 (1995)).

TABLE 1 Alternative Defining Example biological name Interpretationphenotype correlates Variable Value of A1 A₁ 0 1 Operation Results ofoperation F3 0 1 Identity Interaction Growth on URA- Proteins touch F4 10 Not No interaction Growth on 5-FOA Interaction disrupted by mutationin one protein, competed by third protein or peptide aptamer

TABLE 2 Alternative name Defining Phenotype Interpretation VariableValue of variable A₁ 0 1 0 1 A₂ 0 0 1 1 Operation Result of operation F21 0 0 0 Nor White on X-Gal and no No interaction growth on URA- or ofBa₁/P₁ or Ba₂/P₁ Growth on 5-FOA F9 0 0 0 1 And, bridging, Blue on X-GalP₁ interacts with A₁ And A₂ and Growth on URA- Ba₁ and Ba₂ F3 0 0 1 0Ls1, Discrimination, White in X-Gal P₁ interacts with (Not A₁) And A₂and Growth on URA- Ba₂ not Ba₁ F5 0 1 0 0 Ls2, Discrimination, Blue onX-Gal and P₁ interacts with A₁ And (Not A₂) no growth on URA or Ba₁ notBa₂ growth on 5-FOA

TABLE 3 Reporter output One bait cells Two bait cells A₁ A₂ A₃ A₁ A₂Ba(X) Ba(Y) Ba(X) Ba₁(X) Ba₂(Y) Physical interpretation Physicalinterpretation P(Z) P(Z) P(Y) P(Z) P(Z) (one-bait and two bait combined)(one-bait data only) 0 0 0 0 0 X, Y, Z do not interact X, Y, Z, do notinteract 0 0 0 0 1 X, Y, Z associate weakly, modified y and/or zinteract X, Y, Z do not imeract 0 0 0 1 0 X, Y, Z associate weakly,modified X and/or Z interact X, Y, Z do not interact 0 0 0 1 1 X, Y, Zform trimer, requires X, Y, and Z X, Y, Z do not interact 0 0 1 0 0 X, Yform dimer X, Y form dimer 0 0 1 0 1 X modifies Y, modified Y binds Z X,Y form dimer 0 0 1 1 0 Y modifies X, modified X binds Z X, Y form dimer0 0 1 1 1 X, Y form dimer, Z binds X/Y dimer to form X/YIZ trimer X, Yform dimer 0 1 0 0 0 X breaks Y/Z dimer Y, Z form dimer 0 1 0 0 1 Y, Zform dimer, Z discriminates between X and Y Y, Z form dimer 0 1 0 1 0 Ymodifies Z, modified Z binds X Y, Z form dimer 0 1 0 1 1 Y, Z formdimer, X binds to Y/Z dimer to form X/Y/Z trimer Y, Z form dimer 1 0 0 00 Y breaks X/Z dimer X, Z form dimer 1 0 0 0 1 X modifies Z, modified Zbinds Y X, Z form dimer 1 0 0 1 0 X, Z form dimer, Z discriminatesbetween X and Y X, Z form dimer 1 0 0 1 1 X, Z form dimer, Y binds toX/Z dimer to form X/Y/Z trimer X, Z form dimer 0 1 1 0 0 X, Y and Y, Zform dimers, X/Y dimer precludes Y binding Z Y, Z and Y, X form dimers 01 1 0 1 X, Y and Y, Z form dimers, Y/Z dimer predudes X binding Y Y, Zand Y, X forms dimers 0 1 1 1 0 Y modifies X, modified X binds Z Y, Zand Y, X forms dimers 0 1 1 1 1 X/Y and Y/Z dimers interact through Y toform X/Y/Z trimer Y, Z and Y, X form dimers 1 0 1 0 0 X, Y and X, Z formdimers, X/Y dimer precludes X binding Z X, Z and X, Y form dimers 1 0 10 1 X modifies Y, modified Y binds Z X, Z and X, Y form dimers 1 0 1 1 0X, Y and X, Z form dimers, X/Z dimer precludes X binding Y X, Z and X, Yform dimers 1 0 1 1 1 X/Z and X/Y dimers interact through X to formX/Y/Z trimer X, Z and X, Y form dimer 1 1 0 0 0 X, Z and Y, Z formdimers, dimers inactivate one another X, Z and Y, Z form dimers 1 1 0 01 X, Z and Y, Z form dimers, Y/Z dimer precludes X binding Z X, Z and Y,Z form dimers 1 1 0 1 0 X, Z and Y, Z form dimers, X/Z dimer precludes Ybinding Z X, Z and Y, Z form dimers 1 1 0 1 1 X, ZandY, Zdimersform X, Zand Y, Z form dimers 1 1 1 0 0 X, Y forms dimer, X/Y dimer inactivates ZX, Y and X, Z and Y, Z forms dimers, X/Y/Z trimer may form 1 1 1 0 1 X,Y and X, Z and Y, Z form dimers, Y modifies X, modified X, Y and X, Z,and Y, Z X poorly binds Z form dimers, X/Y/Z trimer may form 1 1 1 1 0X, Y and X, Z and Y, Z form dimers, X modifies Y, modified X, Y, and X,Z and Y, Z, Y poorly binds Z form dimers, X/Y/Z trimer may form 1 1 1 11 X, Y and X, Z and Y, Z form dimers, X/Y/Z trimer may form X, Y and X,Z and Y, Z form dimers, X/Y/Z trimer may form

Other Embodiments

In an alternative to the above methods, the systems involving tworeporter genes described herein may be used to select or screen forproteins that bind to two different protein binding sites. To carry outthese screens, two different DNA fragments are inserted upstream of thetwo reporter genes (for example, a URA3 and a lacZ gene), and expressionof each of the genes is assayed in a cell that also contains a geneexpressing a candidate binding protein. This candidate binding proteinmay be expressed in its native state or as a fusion protein joined to anactivation domain.

Any of the methods described herein may be used to screen, withoutlimitation, proteins, peptides, or aptamers.

Other embodiments are within the claims.

16 1 14 DNA Saccharomyces cerevisiae 1 gatccgcggc cgcg 14 2 41 DNASaccharomyces cerevisiae 2 aaaaggaaaa gcggccgctt agttttgctg gccgcatctt c41 3 32 DNA Saccharomyces cerevisiae 3 cggaattctt tcgaaagcta catataaggaac 32 4 56 DNA Escherichia coli 4 tcgagcactc cctatcagtg atagagaaaacactccctat cagtgataga gaaaag 56 5 56 DNA Escherichia coli 5 cgtgagggatagtcactatc tcttttgtga gggatagtca ctatctcttt tcagct 56 6 14 DNASaccharomyces cerevisiae 6 ggccgcggta ccgc 14 7 29 DNA Homo sapiens 7gcctgaattc atgacggaat ataagctgg 29 8 35 DNA Homo sapiens 8 cccgaactcgagtcaggaga gcactgcctt gcagc 35 9 25 DNA Homo sapiens 9 gcctgaattcaaagcaggaa ctgtt 25 10 33 DNA Homo sapiens 10 cccgaactcg agctatcgtggttctatttc tag 33 11 29 DNA Saccharomyces cerevisiae 11 gcctgaattcatgtcttcgg tctcctcag 29 12 33 DNA Saccharomyces cerevisiae 12 cccgaactcgagttatcgaa ataacctaga agg 33 13 31 DNA Homo sapiens 13 gcctgaattcatgaaggggt ggtatcacgg a 31 14 35 DNA Homo sapiens 14 cccgaactcgagctacttga catcattggt ttttg 35 15 20 PRT Homo sapiens 15 Asp Met Asp TrpPhe Phe Arg Phe Tyr Ala Ser Val Ser Arg Leu Phe 1 5 10 15 Arg His LeuHis 20 16 20 PRT Homo sapiens 16 Phe Trp Gln Ala Thr Leu Arg Leu Val SerAsp Lys Leu Ile Leu Leu 1 5 10 15 Tyr Pro Asp Pro 20

What is claimed is:
 1. A method for detecting a protein—proteininteraction, comprising: (a) providing a host cell which contains (i) afirst reporter gene operably linked to a DNA sequence comprising a firstprotein binding site; (ii) a second reporter gene operably linked to aDNA sequence comprising a second protein binding site; (iii) a firstfusion gene which expresses a first fusion protein, said first fusionprotein comprising a first protein covalently bonded to a binding moietywhich is capable of specifically binding to said first protein bindingsite; (iv) a second fusion gene which expresses a second fusion protein,said second fusion protein comprising a second protein covalently bondedto a binding moiety which is capable of specifically binding to saidsecond protein binding site; and (v) a third fusion gene which expressesa third fusion protein, said third fusion protein comprising a thirdprotein covalently bonded to a gene activating moiety; (b) measuringexpression output of said first reporter gene as a measure of saidinteraction between said first and said third proteins; (c) measuringexpression output of said second reporter gene as a measure of saidinteraction between said second and said third proteins; (d)interpreting the expression output results of step (b) and step (c),whereby (i) increased output of both of said first and said secondreporter genes indicates that said third fusion protein interacts withboth of said first and said second fusion proteins; (ii) increasedoutput of said first reporter gene but not said second reporter geneindicates that said third fusion protein interacts with said firstfusion protein but not said second fusion protein; (iii) increasedoutput of said second reporter gene but not said first reporter geneindicates that said third fusion protein interacts with said secondfusion protein but not said first fusion protein; and (iv) no change inoutput in either of said first or said second reporter genes indicatesthat said third fusion protein does not interact with either of saidfirst or said second fusion genes; (e) comparing said expression outputresults of step (b) and step (c) with the expression output resultmeasured in either (i) a first comparison host cell which contains (a) areporter gene operably linked to a DNA sequence comprising a proteinbinding site; (b) a first fusion gene which expresses a first fusionprotein, said first fusion protein comprising said first proteincovalently bonded to a binding moiety which is capable of specificallybinding to said protein binding site; and (c) a second fusion gene whichexpresses a second fusion protein, said second fusion protein comprisingsaid third protein covalently bonded to a gene activating moiety; or(ii) a second comparison host cell which contains (a) a reporter geneoperably linked to a DNA sequence comprising a protein binding site; (b)a first fusion gene which expresses a first fusion protein, said firstfusion protein comprising said second protein covalently bonded to abinding moiety which is capable of specifically binding to said proteinbinding site; and (c) a second fusion gene which expresses a secondfusion protein, said second fusion protein comprising said third proteincovalently bonded to a gene activating moiety; or (iii) both of saidfirst and said second comparison host cells.
 2. The method of claim 1,wherein at least one of said first or said second reporter genes may bereduced in expression level.
 3. The method of claim 1, wherein one ofsaid first or said second protein binding sites is a tetracyclineoperator.
 4. The method of claim 1, wherein one of said first or saidsecond reporter genes is URA3 or lacZ.
 5. The method of claim 1, whereinone of said first or said second reporter genes produces a signal thatis received and detected by a second cell.
 6. The method of claim 1,wherein said host cell is a yeast cell or a mammalian cell.
 7. Themethod of claim 6, wherein said host cell is a yeast cell.
 8. The methodof claim 1, wherein said first and said second reporter genes may beexpressed simultaneously.
 9. The method of claim 1, wherein said firstprotein and said second proteins are allelic variants.
 10. A method fordetecting a protein that mediates a change in the state of anotherprotein, comprising: (a) providing a host cell which contains (i) areporter gene operably linked to a DNA sequence comprising a proteinbinding site; (ii) a first fusion gene which expresses a first fusionprotein, said first fusion protein comprising a first protein covalentlybonded to a binding moiety which is capable of specifically binding tosaid protein binding site; and (iii) a second fusion gene whichexpresses a second fusion protein, said second fusion protein comprisinga second protein which is capable of interacting with said first proteinand which is covalently bonded to a gene activating moiety, wherein atleast one of said first or said second proteins may exhibit a change instate; (b) allowing said first and said second proteins to interact; (c)measuring expression of said reporter gene as a measure of saidinteraction between said first and said second proteins; (d) introducinginto said cell a third gene expressing a third protein; (e) measuringexpression of said reporter gene, a change in said reporter geneexpression in the presence of said third protein being an indicationthat said third protein mediates a change in the state of said first orsaid second protein leading to an alteration in the ability of saidfirst protein and said second protein to interact.
 11. The method ofclaim 10, wherein said change in state is a conformational change. 12.The method of claim 11, wherein said protein exhibiting a conformationalchange is a Ras protein.
 13. The method of claim 10, wherein said hostcell is a yeast cell or a mammalian cell.
 14. The method of claim 13,wherein said host cell is a yeast cell.
 15. The method of claim 10,wherein the expression of each of said first fusion protein and saidthird protein occurs in response to an extracellular stimulus.
 16. Themethod of claim 10, wherein said reporter gene produces a signal that isreceived and detected by a second cell.
 17. A method for detecting aprotein that mediates a change in the state of another protein,comprising: (a) providing a first host cell which contains (i) areporter gene operably linked to a DNA sequence comprising a proteinbinding site; (ii) a first fusion gene which expresses a first fusionprotein, said first fusion protein comprising a first protein covalentlybonded to a binding moiety which is capable of specifically binding tosaid first protein binding site; and (iii) a second fusion gene whichexpresses a second fusion protein, said second fusion protein comprisinga second protein which is capable of interacting with said first proteinand which is covalently bonded to a gene activating moiety, wherein atleast one of said first or said second proteins may exhibit changes instate; (b) allowing said first and said second proteins to interact; (c)measuring expression of said reporter gene in said first host cell as ameasure of said interaction between said first and said second proteins;(d) providing a second host cell which contains (i) said reporter geneoperably linked to said DNA sequence comprising said protein bindingsite; (ii) said first fusion gene which expresses said first fusionprotein; and (iii) said second fusion gene which expresses said secondfusion protein; (iv) a third gene which expresses a third protein; (e)allowing said first and said second proteins to interact in the presenceof said third protein; (f) measuring expression of said reporter gene insaid second host cell as a measure of said interaction between saidfirst and said second proteins in the presence of said third protein, achange in said reporter gene expression in said second host cell ascompared to that measured in said first host cell being an indicationthat said third protein mediates a change in the state of said first orsaid second protein resulting in an alteration in the ability of saidfirst protein and said second protein to interact.
 18. The method ofclaim 17, wherein said change in state is a conformational change. 19.The method of claim 18, wherein said protein exhibiting a conformationalchange is a Ras protein.
 20. The method of claim 17, wherein each ofsaid host cells is a yeast cell or a mammalian cell.
 21. The method ofclaim 20, wherein each of said host cells is a yeast cell.
 22. Themethod of claim 17, wherein the expression of each of said first fusionprotein and said third protein occurs in response to an extracellularstimulus.
 23. The method of claim 17, wherein said reporter geneproduces a signal that is received and detected by a second cell.
 24. Amethod for detecting whether a candidate protein interacts with atranscriptional activator, comprising: (a) providing a host cell whichcontains (i) a reporter gene which can be reduced in expression level,said reporter gene being operably linked to a DNA sequence comprising aprotein binding site; (iii) a first fusion gene which expresses a firstfusion protein, said first fusion protein comprising saidtranscriptional activator covalently bonded to a binding moiety which iscapable of specifically binding to said protein binding site; and (v) asecond fusion gene which expresses a second fusion protein, said secondfusion protein comprising said candidate protein covalently bonded to agene activating moiety; (b) detecting an increase in expression of saidreporter gene as an indication of an interaction between said candidateprotein and said transcriptional activator.
 25. The method of claim 24,wherein said reporter gene is a URA3 gene.
 26. The method of claim 25,wherein said expression level is reduced by 6-azauracil.
 27. The methodof claim 24, wherein said protein binding site is a tetracyclineoperator.
 28. The method of claim 27, wherein said expression level isreduced by tetracycline or a tetracycline derivative.
 29. The method ofclaim 24, wherein said host cell is a yeast cell or a mammalian cell.30. The method of claim 29, wherein said host cell is a yeast cell. 31.A method for detecting a protein—protein interaction, comprising: (a)providing a host cell which contains (i) a first reporter gene operablylinked to a DNA sequence comprising a first protein binding site; (ii) asecond reporter gene operably linked to a DNA sequence comprising asecond protein binding site, wherein one of said first or said secondreporter genes produces a signal that is received and detected by asecond cell; (iii) a first fusion gene which expresses a first fusionprotein, said first fusion protein comprising a first protein covalentlybonded to a binding moiety which is capable of specifically binding tosaid first protein binding site; (iv) a second fusion gene whichexpresses a second fusion protein, said second fusion protein comprisinga second protein covalently bonded to a binding moiety which is capableof specifically binding to said second protein binding site; and (v) athird fusion gene which expresses a third fusion protein, said thirdfusion protein comprising a third protein covalently bonded to a geneactivating moiety; (b) measuring expression output of said firstreporter gene as a measure of said interaction between said first andsaid third proteins; (c) measuring expression output of said secondreporter gene as a measure of said interaction between said second andsaid third proteins; and (d) interpreting the expression output resultsof step (b) and step (c), whereby (i) increased output of both of saidfirst and said second reporter genes indicates that said third fusionprotein interacts with both of said first and said second fusionproteins; (ii) increased output of said first reporter gene but not saidsecond reporter gene indicates that said third fusion protein interactswith said first fusion protein but not said second fusion protein; (iii)increased output of said second reporter gene but not said firstreporter gene indicates that said third fusion protein interacts withsaid second fusion protein but not said first fusion protein; and (iv)no change in output in either of said first or said second reportergenes indicates that said third fusion protein does not interact witheither of said first or said second fusion genes.
 32. The method ofclaim 31, further comprising comparing said expression output results ofstep (b) and step (c) with the expression output result measured ineither (a) a first comparison host cell which contains (i) a reportergene operably linked to a DNA sequence comprising a protein bindingsite; (ii) a first fusion gene which expresses a first fusion protein,said first fusion protein comprising said first protein covalentlybonded to a binding moiety which is capable of specifically binding tosaid protein binding site; and (iii) a second fusion gene whichexpresses a second fusion protein, said second fusion protein comprisingsaid third protein covalently bonded to a gene activating moiety; or (b)a second comparison host cell which contains (i) a reporter geneoperably linked to a DNA sequence comprising a protein binding site;(ii) a first fusion gene which expresses a first fusion protein, saidfirst fusion protein comprising said second protein covalently bonded toa binding moiety which is capable of specifically binding to saidprotein binding site; and (iii) a second fusion gene which expresses asecond fusion protein, said second fusion protein comprising said thirdprotein covalently bonded to a gene activating moiety; or (c) both ofsaid first and said second comparison host cells.
 33. The method ofclaim 31, wherein at least one of said first or said second reportergenes may be reduced in expression level.
 34. The method of claim 31,wherein one of said first or said second protein binding sites is atetracycline operator.
 35. The method of claim 31, wherein one of saidfirst or said second reporter genes is URA3 or lacZ.
 36. The method ofclaim 31, wherein said host cell is a yeast cell or a mammalian cell.37. The method of claim 36, wherein said host cell is a yeast cell. 38.The method of claim 31, wherein said first and said second reportergenes may be expressed simultaneously.
 39. The method of claim 31,wherein said first protein and said second proteins are allelicvariants.